Anti-Viral Treatment With Pertussis Toxin B Oligomer

ABSTRACT

There is disclosed a method for anti-viral therapy, and for decreasing infectivity of viruses that use the chemokine CCR5 receptor as a co-receptor by treatment with the  B. pertussis  toxin (PTX) B oligomer, wherein the PTX B oligomer is composed of from two to ten subunits of PTX B oligomer selected from the group consisting of S2, S3, S4, S5, and combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of, and claims priority from, U.S. patent application Ser. No. 08/911,879, filed Aug. 15, 1997.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention provides methods for anti-viral infection prevention and therapy, and for desensitizing the CCR5 chemokine receptor by treatment with the B. pertussis toxin (PTX) B oligomer.

BACKGROUND OF THE INVENTION

[0003] Viral infections are a continuing medical problem because, like any rapidly-dividing infectious agent, there are continuing mutations that help some sub-populations of viruses resistant to current treatment regimens. Many virally-based diseases do not have effective anti-viral treatments, because such treatments address the symptoms of the viral disease and not the root cause of the disease.

[0004] Pertussis toxin (PTX) is a 105,700 Dalton polypeptide that comprises both an A protomer and a B oligomer (PTX B oligomer). Pertussis toxin, a heterohexameric protein released by Bordetella pertussis, exhibits diverse biological activities, mediated mostly by the A-subunit (A-protomer) which inactivates signaling pathways of members of the G_(i)-G_(o) and G_(t)-protein family. Binding to the receptor and internalization of the toxin is mediated by the B-oligomer. The hexamer is composed of one S1 subunit having a molecular weight of 28 kDa, one S2 subunit having a molecular weight of 23 kDa, one S3 subunit having a molecular weight of 22 kDa, two S4 subunits having a molecular weight of 11.7 kDa, and one S5 subunit having a molecular weight of 9.3 kDa. The S1 subunit constitutes the active A-protomer with ADP-ribosyltransferase activity. The B-oligomer is a pentameric protein complex composed of two dimers (S2-S4 and S3-S4) joined together by the S5 subunit, and is responsible for target cell binding. (Ui, Pertussis Toxin as a Probe of Receptor Coupling to Inositol Lipid Metabolism. Phosphoinositides and Receptor Mechanism, pp 163-195. Alan R. Liss, Inc., 1986; Wong et al., “Pharmacology of pertussis toxin B-oligomer” Can. J Physiol. Pharmacol. 74:559-564, 1996). In addition, the D1 oligomer is composed of one each of the S2 and S4 subunits and can bind to a p43 PTX receptor, and a D2-oligomer is composed of one each of the S3 and S4 subunits and can bind to a p70 PTX receptor (Wong and Rosoff, “Pharmacology of Pertussis Toxin B” Can. J Physiol. Pharmacol. 74:559-566, 1996).

[0005] The A-protomer is released from the holotoxin molecule as a result of an allosteric effect of intracellular ATP. Specifically, intracellular ATP binds to the S3 subunit of the B-oligomer. The active center of ADP-rybosyltransferase activity, unmasked in the released A-promoter molecule, can interact with intracellular reduced glutathione, which cleaves disulfide bonds essential for enzymatic activity (Ui, Pertussis Toxin as a Probe of Receptor Coupling to Inositol Lipid Metabolism. Phosphoinositides and Receptor Mechanism, pp 163-195. Alan R. Liss, Inc., 1986). The A-subunit possesses adenine diphosphate (ADP) ribosyltransferase activity, which catalyzes ADP-ribosylation of G-proteins, leading to their dissociation from receptors and uncoupling of associated signal transduction events. Due to this feature, PTX has become a very useful pharmacological tool for the identification of G proteins in the plasma membrane.

[0006] The PTX B oligomer (binding) confers cell membrane-binding specificity by interacting with specific receptors. In lymphocytes, two PTX-binding proteins have been identified: a 43-kDa (Rogers et al., J. Immunol. 145:678-683, 1990) and a 70-kDa (Armstrong et al., Infect. Immun. 62:2236-2243, 1994) receptors. A leukocyte-specific integrin, Mac-1 (CD11b/CD18) may be a binding site for PTX on macrophages (Wong et al., Immunology 88:90-97, 1996). Occupation of these putative receptors by the PTX B oligomer can trigger phospholipase C (PLC) and tyrosine kinase-dependent signal transduction pathways. However, the effect of these events on the function of a target cell is not characterized, and pharmacological properties of the PTX B-oligomer are largely unknown. Nevertheless, the PTX B oligomer was shown to potentiate the immune response to intranasally administered influenza vaccine in mice when used as an adjuvant (Oka et al., Vaccine 12:1255-1258, 1994), and also induced resistance to lethal doses of mouse adenovirus infection (Winters et al., Dev. Biol. Stand. 61:233-240, 1985). The B-oligomer was shown to improve immune responses to viral vaccines, when used as an adjuvant (Oka et al., Vaccine 12:1255-1258, 1994; and Winters et al., Dev. Biol. Stand. 61:233-240,1985).

[0007] In addition, whole B. pertussis Toxin (PTX) affected HIV replication in U1 cells (in vitro) wherein there was demonstrated a role of Gi protein PTX sensitivity in the U1 chronically infected monocytic cell line (Chowdury et al., Virology 203:378-383, 1994). In addition the stimulated PTX receptor can induce phospholipase C, which cuts off PIP₂ and produces IP3 (inositol triphosphate) and DAG (diacyl glycerol) (Rosoff and Mohan, J Immunol. 149:3191-3199, 1992). The PTX receptor further appears to require the coexpression of a CD3/TCR complex (Gray et al., J. Immunol. 142:1631-1638, 1989). Moreover, concentrations of the B oligomer of PTX (100 nM) stimulated production of interleukin-2 (IL-2) with a similar pattern seen with the antibody OKT3 in vitro in Jurkat cells (Rosoff et al., J Immunol. 139:2419-2423, 1987).

[0008] Anti-viral therapies directed against the virus (as opposed to directed to symptoms of the disease) have generally been based upon viral enzymatic inhibition, such as HIV therapies directed against viral reverse transcriptase or viral protease enzymes. Therefore, there is a need in the art to discover and develop new anti-viral therapies that are not based upon a mechanism of action to inhibit virus-specific enzyme that is used in viral replication.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method for treating viral infections, comprising administering an effective amount of B. pertussis PTX B oligomer to a patient having a viral infection. Preferably, the daily dose administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the viral infection is caused by HIV. Preferably, the PTX B oligomer is composed of combinations of from two to ten subunits of PTX B oligomer selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4

[0010] The present invention further provides a method for treating viral infections, comprising administering an effective amount of B. pertussis toxin PTX B oligomer to a patient having a viral infection, wherein the viral infection is inhibited by a mechanism selected from the group consisting of inhibition of viral entry into the cell, inhibition of post-entry replication, inhibition of co-capping of a viral receptor or co-receptor, desensitization of a viral receptor or co-receptor, modulation of protein kinase C (PKC) activity, and combinations thereof. Preferably, the daily dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the viral infection is caused by a retrovirus, such as HIV. Preferably, the PTX B oligomer comprises combinations of from two to ten subunits of PTX B oligomer selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0011] The present invention further provides a method for decreasing the infectivity of a cell or susceptibility of a cell to retroviral infection, wherein the cell expresses a CCR5 chemokine receptor, and wherein a virus uses the CCR5 as a co-receptor, comprising contacting the cell with an amount of B. pertussis PTX B oligomer sufficient to induce an antiviral mechanism selected from the group consisting of inhibition of viral entry into the cell, inhibition of post-entry replication, inhibition of co-capping of a viral receptor or co-receptor, desensitization of a viral receptor or co-receptor, modulation of protein kinase C (PKC) activity, and combinations thereof. Preferably, the viral infection is caused by a retrovirus, such as HIV. Preferably, the PTX B oligomer comprises combinations of from two to ten subunits of PTX B oligomer, wherein the subunit of PTX B oligomer is selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0012] The present invention further provides a method for treating a CCR5 receptor-related physiological or pathological condition, comprising administering to a patient having the CCR5 receptor-related physiological or pathological condition, an amount of B. pertussis toxin PTX B oligomer sufficient to desensitize the CCR5 receptor, wherein the PTX B oligomer comprises from two to ten subunits of PTX B oligomer, wherein the subunit of PTX B oligomer is selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. Preferably, the CCR5 receptor-related physiological or pathological condition is selected from the group consisting of progressive neurological disorders, HTLV-associated myelopathy, multiple sclerosis, inflammation, and infections.

[0013] The present invention further provides an anti-HIV vaccine, comprising a HIV antigen or antigens and PTX B oligomer as a HIV vaccine adjuvant, wherein the PTX B oligomer is composed of from two to ten subunits of PTX B oligomer selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.In addition, the present invention provides a method of treating HIV infection, comprising administering an effective amount of an anti-HIV vaccine, wherein the anti-HIV vaccine comprises a HIV antigen or antigens and PTX B oligomer as a HIV vaccine adjuvant, wherein the PTX B oligomer is composed of from two to ten subunits of P PTX B oligomer TX selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0014] The present invention provides vaccine formulation for vaccinating against opportunistic infections from non-HIV pathogens in HIV-infected individuals, consisting essentially of an HIV-suppressing formulation of B. pertussis toxin (PTX) B oligomer (PTX B oligomer), an antigenic component specific for the non-HIV pathogen, with the proviso that the non-HIV pathogen is not influenza, and a vaccine-acceptable carrier. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the pathogen is selected from the group consisting of Pneumocistis, Candida, CMV (Cytomegalovirus), Hepatitis virus (A, B and C), Pneumococcus, Mycobacterium tuberculosis, Mycobacterium aviium, Cryptosporidium, and Aspergillis. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0015] The present invention further provides a method for vaccinating against opportunistic infections from non-HIV pathogens in HIV-infected individuals, comprising administering an HIV-suppressing formulation of B. pertussis toxin (PTX) B oligomer (PTX B oligomer) and an antigenic component specific for the non-HIV pathogen. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the pathogen is of bacterial, viral, fungal, parasitic, or mycobacterial origin. Preferably, the pathogen is selected from the group consisting of Pneumocistis, Candida, CMV (Cytomegalovirus), Hepatitis virus (A, B and C), Pneumococcus, Mycobacterium tuberculosis, Mycobacterium aviium, Cryptosporidium, and Aspergillis. Preferably the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0016] The present invention further provides a method for vaccinating to protect an individual against HIV infection in an individual not infected with HIV comprising administering a vaccine composition comprising B. pertussis toxin (PTX) B oligomer (PTX B oligomer) and an antigenic component specific for HIV. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0017] The present invention further provides an HIV preventive vaccine formulation consisting essentially of B. pertussis toxin B oligomer (PTX B oligomer), an antigenic component specific for HIV, and a vaccine-acceptable carrier. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0018] The present invention further provides a method for treating HIV infection, comprising administering B. pertussis toxin B oligomer (PTX B oligomer) in combination with an anti-HIV agent, wherein the HIV agent is selected from the group consisting of protease inhibitors, reverse transcriptase inhibitors, nuclear localization importation inhibitors, and combinations thereof Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0019] The present invention further provides a method for preventing HIV infection in an individual recently having transmissable contact with an HIV-infected individual, comprising administering B. pertussis toxin (PTX) B oligomer (PTX B oligomer) in combination with an agent selected from the group consisting a nuclear localization importation inhibitor, reverse transcriptase inhibitors, protease inhibitors, and combinations thereof. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0020] The present invention further provides a pharmaceutical composition for treating viral infections consisting essentially of PTX B oligomer and a pharmaceutically acceptable carrier. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIGS. 1 and 2 provide the results of a study showing the inhibitory effects of PTX B oligomer on HIV-1 replication in primary T cells. Specifically, FIG. 1 shows the results when primary T cells were infected with NSI strain of HIV (92U660 a macrophage-tropic primary HIV-1 isolate) and either 1 nM or 0.1 nM of PTX B oligomer (specifically, 1S1-1S2-1S3-2S4-1S5) was added to the cultures. Cell proliferation was measured by tritiated thymidine uptake (left panel) and anti-viral activity was measured by RT (reverse transcriptase) activity in culture supernatants collected every 3 days (right panel). FIG. 2 shows the results when primary T cells were infected with SI strain of HIV-1_(LAI) (a T cell tropic HIV-1 strain) and either 1 nM or 0.1 nM of PTX B oligomer (specifically, 1S2-1S3-2S4-1S5) was added to the cultures. Cell proliferation was measured by tritiated thymidine uptake (left panel) and anti-viral activity was measured by RT (reverse transcriptase) activity in culture supernatants collected every 3 days (right panel). These data show anti-viral activity of PTX B oligomer in a dose response fashion in this predictive assay.

[0022]FIG. 3 shows that pretreatment of T lymphocytes with PTX B oligomer induced production of β-chemokines (MIP-1α, MIP-1β and RANTES) and their levels correlated with suppression of viral replication. Specifically, FIG. 3 shows a comparison of RT inhibition in culture supernatants measured as a ratio of RT activity in cells (92US660 in the left panel and HIV-1_(LAI) in the right panel) pre-treated with different concentrations of PTX B oligomer (specifically, 1S2-1S3-2S4-1S5) and untreated cells. RT activity was tested at the peak of viral infection on day 9 and MIP-1α was tested in the same culture supernatants by ELISA.

[0023]FIG. 4 (top panel) shows a comparison of hours of pretreatment with PTX B oligomer (specifically, 1S2-1S3-2S4-1S5) (0.1 nM) demonstrating that 2 hours pretreatment is enough to increase levels of β-chemokines in normal primary T cells. Similarly, the bottom panel shows that PTX B oligomer (specifically, 1S2-1S3-2S4-1S5) can influence an already-established HIV-1 infection, since adding 3 days after infection can still decrease RT activity.

[0024]FIG. 5 shows that PTX B oligomer is able to induce up-regulation of β-chemokines when added once immediately after infection, without any difference versus the condition with all time treatment. Specifically, FIG. 5 shows the effect of 18 hours of pre-treatment with PTX B oligomer (specifically, 1S2-1S3-2S4-1S5) (0.1 nM) all time after infection or one time (1t) on RANTES production (measured by ELISA) in primary T cells infected by T cell line tropic (LAI) HIV-1 strains.

[0025]FIG. 6 shows the effect of PTX and PTX B oligomer on HIV-1 entry into cells. Specifically, in panel A, monocyte-depleted PBMC cultures were treated with PTX (1 nM) or left untreated (control), and inoculated with R5 HIV-1_(92US660) or X4 HIV-1_(LAI). In panel B, cells were treated with the indicated concentrations of PTX B oligomer or an anti-CD4 MAb Leu3A for 10 min. or left untreated and inoculated with HIV-1_(92US660) (left panel) or HIV-1_(LAI) (middle panel).

[0026]FIG. 7 shows that PTX B oligomer inhibits HIV-1 replication. Specifically, in panels A, B, and C, monocyte-depleted PBMCs from HIV seronegative donors or a PM1 T-cell line (insets) were pretreated for 1 hr with 1 nM of PTX B oligomer and infected with primary R5 (92US660) or X4 (92UG21) strains, or T cell line-adapted X4 virus LAI, respectively. Subsequent culturing was in the presence of 1 nM of PTX B oligomer. Panel D shows direct analysis of clinical isolates. Panel E shows the uptake of [³H]thymidine over time.

[0027]FIG. 8 shows that PTX B oligomer does not block ligand binding to CCR5. Analysis of [¹²⁵I]MIP-β binding to untreated or PTX B oligomer-treated PBMCs was performed as described in Example 8 “Methods”. Binding was also measured in the presence of either a 1000-fold molar excess of unlabeled (cold) MIP-1β, or 0.5 μg/ml of gp120 of an R5 (JR-FL) or X4 (LAI) HIV-1 strain (panel A). To control for non-specific effects of the PTX B oligomer, binding was also analyzed using cells treated either with PTX B oligomer (1 nM) at 4° C., or with 75 ng/ml of BSA at 37° C. (panel B).

[0028]FIG. 9 shows an analysis of cross-desensitization of chemokine and PTX receptors. Fura-2-loaded PBMC were stimulated with 0.83 tg/ml of PTX B oligomer (B-ol.), (panels A and B), 1.66 μg/ml of MIP-1β (panel C), or 0.1 μg/ml of SDF-1α (panel D) at the time indicated by the left arrow. At the time indicated by the right arrow (3-5 min. later), cells were rechallenged with PTX B oligomer (panels C, D), MIP-1β (panel A), or SDF-1α (panel B) at the same concentration as in the first challenge.

[0029]FIG. 10 shows that the effect of PTX B oligomer on CCR5 is reversed by PKC inhibitor. Panel A shows that Ro-31-8220 reversed the inhibitory effect of PTX B oligomer on entry of R5 strains. Panel B shows that Ro-31-8220 reversed the inhibitory effect of PTX B oligomer on signaling from CCR5.

[0030]FIG. 11 shows an analysis of receptor capping on PBMCs by immunofluorescent microscopy. Co-localization of chemokine receptors and CD4 resulted in overlapping of red and green fluorescence, thus producing yellow color. Approximately 50% and 30% of CD4/CCR5 and CD4/CXCR4 double-positive cells, respectively, exhibited capping.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Examples of the present invention demonstrate that treatment of primary T cells with PTX B-oligomer not only blocks replication of both R5 and X4 HIV strains, but also induces a specific desensitization of the chemokine receptor CCR5. As a result, such cells do not respond to stimulation with a CCR5 ligand, MIP-1β, do not support co-capping of CCR5 and CD4, and do not support entry of CCR5-dependent HIV-1 strains into cells.

[0032] Anti-Viral Method of Treatment

[0033] The present invention provides a method for treating viral infections, comprising administering an effective amount of Pertussis toxin PTX B oligomer to a patient having a viral infection, wherein the PTX B oligomer is composed of from two to ten subunits of PTX B oligomer selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, the daily dose administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the viral infection is caused by HIV.

[0034] The present invention further provides a method for treating viral infections, comprising administering an effective amount of B. pertussis toxin B oligomer (PTX B oligomer) to a patient having a viral infection, wherein the viral infection is inhibited by a mechanism selected from the group consisting of inhibition of viral entry into the cell, inhibition of post-entry replication, inhibition of co-capping of a viral receptor or co-receptor, desensitization of a viral receptor or co-receptor, modulation of protein kinase C (PKC) activity, and combinations thereof Preferably, the daily dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the viral infection is caused by a retrovirus, such as HIV. Preferably, the PTX B oligomer comprises combinations of from two to ten subunits of PTX B oligomer selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0035] The present invention further provides a method for decreasing the infectivity of a cell or susceptibility of a cell to retroviral infection, wherein the cell expresses a CCR5 chemokine receptor, and wherein a virus uses the CCR5 as a co-receptor, comprising contacting the cell with an amount of B. pertussis toxin (PTX) PTX B oligomer sufficient to induce an antiviral mechanism selected from the group consisting of inhibition of viral entry into the cell, inhibition of post-entry replication, inhibition of co-capping of a viral receptor or co-receptor, desensitization of a viral receptor or co-receptor, modulation of protein kinase C (PKC) activity, and combinations thereof. Preferably, the viral infection is caused by a retrovirus, such as HIV. Preferably, the PTX B oligomer comprises combinations of from two to ten subunits of PTX B oligomer, wherein the PTX B oligomer is selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

[0036] The present invention further provides a method of treating a CCR5 receptor-related physiological or pathological condition, comprising administering to a patient having the CCR5 receptor-related physiological or pathological condition, an amount of B. pertussis toxin (PTX) B oligomer sufficient to desensitize the CCR5 receptor, wherein the PTX B oligomer comprises from two to ten subunits of PTX B oligomer, wherein the subunit of PTX B oligomer is selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. Preferably, the CCR5 receptor-related physiological or pathological condition is selected from the group consisting of progressive neurological disorders, HTLV-associated myelopathy, multiple sclerosis, inflammation, and infection.

[0037] Specifically, the present invention provides for specific desensitization of CCR5 of primary T cells by treatment with PTX B-oligomer. As a result, such cells do not respond to stimulation with a CCR5 ligand, MIP-1β, do not support co-capping with CD4, and do not support entry of CCR5-dependent HIV-1 strains. The data provided in predictive assays of anti-viral activity are reasonably correlated to the claimed method of use.

[0038] PTX B Oligomer

[0039] PTX B oligomer (specifically, 1S2-1S3-2S4-1S5 PTX B oligomer) is available commercially (e.g., Sigma or Calbiochem) in laboratory reagent quantities. PTX B oligomer, according to the present invention, is composed of from two to ten subunits wherein the subunits are S2, S3, S4, and S5. The sequence characterization and molecular weights of each subunit is known in the art (see, for example, Tamura et al., Biochemistry 21:5516-5522, 1982; and Nicosia et al., Proc. Natl. Acad. Sci. USA 83:4631-4635, 1986). The designation “2S2-1S3” for example refers to an oligomer composed of two S2 subunits and one S3 subunit. Similarly, the preferred 1S1-1S2-1S3-2S4-1S5 PTX B oligomer is a six subunit oligomer having one S1, one S2, one S3, two S4s and one S5 subunits.

[0040] Pharmaceutical or Vaccine Formulation

[0041] The inventive method in the form of a pharmaceutical composition comprising PTX B oligomer can be administered to a patient either by itself (complex or combination) or in pharmaceutical compositions where it is mixed with suitable carriers and excipients. A vaccine formulation is generally a pharmaceutical formulation for injection (subcutaneous or intramuscular) that further contains an adjuvant. PTX B oligomer can be administered parenterally, such as by intravenous injection or infusion, intraperitoneal injection, subcutaneous injection, or intramuscular injection. PTX B oligomer can be administered orally or rectally through appropriate formulation with carriers and excipients to form tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like. PTX B oligomer can be administered topically, such as by skin patch, to achieve consistent systemic levels of active agent. PTX B oligomer is formulated into topical creams, skin or mucosal patch, liquids or gels suitable to topical application to skin or mucosal membrane surfaces. PTX B oligomer can be administered by inhaler to the respiratory tract for local or systemic treatment of HIV infection.

[0042] The dosage of PTX B oligomer suitable for use with the present invention can be determined by those skilled in the art from this disclosure. PTX B oligomer will contain an effective dosage (depending upon the route of administration and pharmacokinetics of the active agent) of PTX B oligomer and suitable pharmaceutical carriers and excipients, which are suitable for the particular route of administration of the formulation (i.e., oral, parenteral, topical or by inhalation). The active PTX B oligomer is mixed into the pharmaceutical formulation by means of mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical formulations for parenteral administration include aqueous solutions of the active PTX B oligomer in water-soluble form. Additionally, suspensions of the active PTX B oligomer may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may optionally contain stabilizers or agents to increase the solubility of the complex or combination to allow for more concentrated solutions.

[0043] Pharmaceutical formulations for oral administration can be obtained by combining the active compound with solid excipients, such as sugars (e.g., lactose, sucrose, mannitol or sorbitol), cellulose preparations (e.g., starch, methyl cellulose, hydroxypropylmethyl cellulose, and sodium carboxymethyl cellulose), gelaten, gums, or polyvinylpyrrolidone. In addition, a desintegrating agent may be added, and a stabilizer may be added.

[0044] Use of PTX B Oligomer as a Vaccine Adjuvant

[0045] No effective vaccination can be achieved without the use of a potent adjuvant. The mechanisms by which some adjuvants, such as alumn or Freud's adjuvant, potentiate immune response to an immunogen is up-regulation of cytokine production by certain target cells (TH1 lymphocytes for alumn and TH2 cells for Freud's adjuvant). PTX B oligomer can be used as an adjuvant for HIV vaccine. The data provided herein show that HIV-1 down-regulates β-chemokine production in infected T lymphocytes, thus reducing the protective effect of this potent anti-HIV mechanism, while PTX B oligomer can negate this HIV action. Therefore, PTX B oligomer stimulates uninfected T cells and restores the compromised capacity of infected T lymphocytes to produce β-chemokines.

[0046] Uses and Compositions Comprising PTX B Oligomer

[0047] The data provided herein provide a reasonable correlation to multiple uses and pharmaceutical composition, including vaccine compositions, for PTX B oligomer alone or in combination with other known anti-HIV agents or with appropriate antigens in vaccine formulations. A vaccine formulation for vaccinating against opportunistic infections from non-HIV pathogens in HIV-infected individuals, consists essentially of an HIV-suppressing formulation of B. pertussis toxin (PTX) B oligomer (PTX B oligomer), an antigenic component specific for the non-HIV pathogen, with the proviso that the non-HIV pathogen is not influenza, and a vaccine-acceptable carrier. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the pathogen is selected from the group consisting of Pneumocistis, Candida, CMV (Cytomegalovirus), Hepatitis virus (A, B and C), Pneumococcus, Mycobacterium tuberculosis, Mycobacterium aviium, Cryptosporidium, and Aspergillis. Other opportunistic pathogens may be involved, but the foregoing lists the most common opportunistic pathogens published to be associated with HIV infected individuals. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. The antigenic component is taken from the path organism, or a synthetic variant thereof. This formulation uses the dual properties of PTX B oligomer to have HIV-suppressing activity and to possess immune-stimulation adjuvant activity to enhance the host immune response to the antigen of the vaccine formulation. Thus, there may or may not even require an additional adjuvant added to the vaccine formulation. This vaccine composition is used for a method for vaccinating against opportunistic infections from non-HIV pathogens in HIV-infected individuals and comprises administering an HIV-suppressing formulation of B. pertussis toxin (PTX) B oligomer (PTX B oligomer) and an antigenic component specific for the non-HIV pathogen.

[0048] The present invention further provides a method for vaccinating to protect an individual against HIV infection in an individual not infected with HIV comprising administering a vaccine composition comprising B. pertussis toxin (PTX) B oligomer (PTX B oligomer) and an antigenic component specific for HIV. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. The data supporting the utility of PTX B oligomer shows that it is useful as a preventive vaccine for both its anti-HIV therapeutic activity and as a vaccine adjuvant. The HIV preventive vaccine formulation consisting essentially of B. pertussis toxin B oligomer (PTX B oligomer), an antigenic component specific for HIV, and a vaccine-acceptable carrier. The HIV-specificity is conferred by the HIV-based antigenic component, such as gp120.

[0049] The present invention further provides a method for treating HIV infection, comprising administering B. pertussis toxin B oligomer (PTX B oligomer) in combination with an anti-HIV agent, wherein the HIV agent is selected from the group consisting of protease inhibitors, reverse transcriptase inhibitors, nuclear localization importation inhibitors, and combinations thereof. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. This method provides that the anti-HIV therapeutic activity of PTX B oligomer is useful both alone and in combination with other known anti-HIV agents. HIV therapeutics are often administered in combinations of different classes of agents, wherein the most common classes are reverse transcriptase inhibitors (such as AZT) and protease inhibitors (several are marketed). In addition, nuclear localization inhibitors are described in U.S. Pat. Nos. 5,849,793; 5,703,086; 5,733,932; 5,808,068, 5,574,040; 5,620,983; and 5,840,893, the disclosures of which are incorporated by reference herein.

[0050] The present invention further provides a method for preventing HIV infection in an individual recently having transmissable contact with an HIV-infected individual, comprising administering B. pertussis toxin (PTX) B oligomer (PTX B oligomer) in combination with an agent selected from the group consisting a nuclear localization importation inhibitor, reverse transcriptase inhibitors, protease inhibitors, and combinations thereof. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. The method is also useful for administration in a “morning after” situation where there was though to have been potential contact with an HIV infected individual (or some other form of exposure likely to result in transmission of the virus) and there is a therapeutic need for administration of a bolus of anti-HIV activity that is dependent on mechanism provided herein to prevent cells being infected by circulating virus. Those agents most likely to act to prevent cell infectivity are PTX B oligomer and nuclear localization inhibitors.

[0051] Lastly, the present invention further provides a pharmaceutical composition for treating viral infections consisting essentially of PTX B oligomer and a pharmaceutically acceptable carrier. Preferably, the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg. Preferably, the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. The data provided herein a show therapeutic activity of PTX B oligomer as much more than an adjunct, but as a therapeutic agent that can be used alone.

[0052] Accordingly, the present invention provides an anti-HIV vaccine, comprising a HIV antigen or antigens and PTX B oligomer as a HIV vaccine adjuvant, wherein the PTX B oligomer is composed of from two to ten subunits of PTX selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, 1S2-1S3-2S4, and 1S2-1S4-1S3-1S4-1S5. In addition, the present invention provides a method of treating HIV infection, comprising administering an effective amount of an anti-HIV vaccine, wherein the anti-HIV vaccine comprises a HIV antigen or antigens and PTX B oligomer as a HIV vaccine adjuvant, wherein the PTX B oligomer is composed of from two to ten subunits of PTX selected from the group consisting of S2, S3, S4, S5, and combinations thereof. Preferably, PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.

EXAMPLE 1

[0053] Materials and Methods

[0054] Reagents. PTX was purchased from Sigma (St. Louis, Mo.) and PTX B oligomer from Calbiochem (La Jolla, Calif.). The purity of PTX B oligomer was verified by gel electrophoresis analysis that revealed lack of contamination with A-protomer. MIP-1β, RANTES, and SDF-1α were from PeproTech (Rocky Hill, N.J.), and Fura-2/AM, Ro-31-8220, and Go 6979 were from Calbiochem.

[0055] Primary lymphocyte cultures. T cell-enriched, monocyte-depleted cultures were established from peripheral blood mononuclear cells from HIV-1-negative donors by Ficoll-Hypaque gradient centrifugation and two rounds of adherence to plastic. Non-adherent cells were collected by centrifugation, resuspended in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), and stimulated with phytohemagglutinin (5 μg/ml) for 3 days. Cells were then washed and cultured for another 7 days in medium supplemented with 20 U/ml of recombinant IL-2. By that time 84% of the cells were CD3⁺, 40% CD4⁺, 30% CCR5⁺, 95% CXCR4⁺, 10% CCR5⁺CD4⁺, and 35% CXCR4⁺CD4⁺, as determined by flow cytometry.

[0056] Infection with HIV-1. Five HIV-1 strains were used in this study: three R5 viruses (92US660, 92US657, ADA) and two X4 strains (92UG21, a primary isolate, and LAI, a T cell line-adapted virus). Prior to infection, viral stocks were treated with 200 U/ml of RNAse-free DNAse for 1 hr at room temperature to eliminate DNA contamination. Viral inoculae were adjusted according to reverse transcriptase activity to 6×10⁴ cpm per 10⁶ cells. After a 2 hr adsorption, cells were washed and cultured in IL-2-supplemented medium.

[0057] This example illustrates an experiment showing the inhibitory effects of PTX B oligomer on HIV-1 replication in primary T cells. Treatment of primary T cells with PTX B oligomer resulted in a complete inhibition of replication of both macrophage-tropic and T cell tropic HIV-1 strains, without affecting T cell proliferation and viability. Specifically, FIG. 1 shows the results when primary T cells were infected with NSI strain of HIV (92U660 a macrophage-tropic primary HIV-1 isolate) and either 1 nM or 0.1 nM of PTX B oligomer was added to the cultures. Cell proliferation was measured by tritiated thymidine uptake (left panel) and anti-viral activity was measured by RT (reverse transcriptase) activity in culture supernatants collected every 3 days (right panel). FIG. 2 shows the results when primary T cells were infected with SI strain of HIV-1_(LAI) (a T cell tropic HIV-1 strain) and either 1 nM or 0.1 nM of PTX B oligomer was added to the cultures. Cell proliferation was measured by tritiated thymidine uptake (left panel) and anti-viral activity was measured by RT (reverse transcriptase) activity in culture supernatants collected every 3 days (right panel). These data show anti-viral activity of PTX B oligomer in a dose response fashion in this predictive assay.

EXAMPLE 2

[0058] This example illustrates results of experiments wherein primary T cell cultures treated with PTX B oligomer displayed increased levels of β-chemokines (MIP-1α, MIP-1β and RANTES), which are potent suppressors of macrophage-tropic strains of HIV-1 in primary T cells. Specifically, FIG. 3 shows that pretreatment of T lymphocytes with PTX B oligomer induces production of β-chemokines (MIP-1α, MIP-1β and RANTES) and their levels correlated with suppression of viral replication. FIG. 3 shows a comparison of RT inhibition in culture supernatants measured as a ratio of RT activity in cells (92US660 in the left panel and HIV-1_(LAI) in the right panel) pre-treated with different concentrations of PTX B oligomer and untreated cells. RT activity was tested at the peak on day 9 and MIP-1α a was tested in the same culture supernatants by ELISA.

EXAMPLE 3

[0059] This example illustrates the PTX B oligomer dosing schedules, as coordinated with infection times, needed to cause an increase in the levels of β-chemokines in normal primary T cells. FIG. 4 (top panel) shows a comparison of hours of pretreatment with PTX B oligomer (0.1 nM) demonstrating that 2 hours pretreatment is enough to increase levels of β-chemokines in normal primary T cells. Similarly, the bottom panel shows that PTX B oligomer can influence an already-established HIV-1 infection, since adding 3 days after infection can still decrease RT activity. FIG. 4 shows the effect of 18 hour (−18h) and 2 hours (−2) of pre-treatment with PTX B oligomer (0.1 nM) on MIP-1αand RT activity. Three day post infection treatment (d3) by PTX B oligomer could still inhibit HIV replication.

EXAMPLE 4

[0060] This example illustrates that PTX B oligomer was able to induce up-regulation of β-chemokines when added once immediately after infection, without any difference versus the condition with all time treatment. Specifically, FIG. 5 shows the effect of 18 hours of pre-treatment with PTX B oligomer (0.1 nM) all time after infection or one time (1t) on RANTES production (measured by ELISA). Both macrophage tropic (92US660) and T cell line tropic (LAI) HIV-1 strains were inhibited.

[0061] β-chemokines can inhibit infection of T lymphocytes with macrophage-tropic, but not with T-cell-line-tropic viruses (Cocchi et al. Science 270:1811-15, 1995). T lymphocytes do not produce SDF-1 (a ligand for the CXCR4 receptor used by T-cell-tropic strains). Therefore, these data suggest that mechanisms other than β-chemokine induction are involved in anti-HIV activity of PTX B oligomer.

EXAMPLE 5

[0062] This example illustrates that pertussis toxin (PTX) inhibited entry of R5 HIV-1 strains, but not of X4 strains. PTX was used to specifically inactivate G₁-like proteins that transduce signals from both CCR5 and CXCR4 (Davis et al., J Exp. Med. 186:1793-1798, 1997). In panel A of FIG. 6, monocyte-depleted PBMC cultures were treated with PTX (1 nM) or left untreated (control), and inoculated with R5 HIV-1_(92US660) or X4 HIV-1_(LAI). Viral entry was analyzed by PCR using primers LTR R/U5 specific for the early RT product, as described previously (Schmidtmayerova et al., J. Virol. 72:4633-4642, 1998). The fragment of HIV-1 cDNA amplified by these primers was produced either within the virion or very early after virus-cell fusion, and reflected the efficiency of virus entry. Results were quantified on a Direct Imager (Packard Instrument Company, Meriden, Conn.) and are presented as a percent of counts in treated versus control samples. The error bars show the deviation from the mean for triplicate samples. Similar results were obtained in two independent experiments with cells from different donors.

[0063] Surprisingly, PTX inhibited entry of R5 HIV-1 strains, 92US660 (FIG. 6A) and ADA, but not of X4 strains, LAI (FIG. 6A) and 92UG21.

EXAMPLE 6

[0064] This example illustrates that the observed inhibitory effect of PTX on entry of R5 HIV was accounted for by the PTX B oligomer, and was independent of inactivation of G_(i)-like proteins by the A-protomer. PTX is a complex protein composed of an active (A) proteome and a binding (B) oligomer. PTX B oligomer was tested to see whether its activity could account for the observed inhibitory effect of PTX on entry of R5 HIV-1. In panel B of FIG. 6, cells were treated with the indicated concentrations of PTX B oligomer or an anti-CD4 MAb Leu3A for 10 min. or left untreated and inoculated with HIV-1_(92US660) (left panel) or HIV-1_(LAI) (middle panel). Viral entry was analyzed as in panel A. In parallel, each sample was amplified with α-tubulin-specific primers to control for the total amount of DNA.

[0065] Similar to results observed with PTX B-oligomer inhibited, in a dose-dependent fashion, entry of R5, but not of X4, HIV-1 strains (FIG. 6B). Analysis of the PTX B oligomer preparation by SDS-PAGE confirmed the lack of A-protomer, and G_(i)-mediated signaling was not impaired in PTX B oligomer-treated cells (FIG. 9, right panel).

[0066] In summary, PTX B oligomer blocked entry of R5 HIV-1 strains by a mechanism that was independent of G₁ protein inhibition.

EXAMPLE 7

[0067] This example illustrates that, in addition to its effect on HIV-1 entry via the CCR5 receptor, PTX B oligomer exerted its anti-HIV activity on both R5 and X4 strains of HIV via a mechanism of inhibiting post-entry replication. The effect of PTX B oligomer on HIV-1 replication in long-term cultures was analyzed by measuring R5 or X4 HIV-1 virus production after in vitro infection of primary PBMCs from HIV-seronegative donors (FIG. 7). For in vitro infection, monocyte-depleted PBMCs from HIV seronegative donors or PM1 T cell lines (insets) were pretreated for 1 hr with 1 mM of PTX B oligomer and infected with primary R5 (92US660, FIG. 7A) or X4 (92UG21, FIG. 7B) strains, or T cell line-adapted X4 virus LAI (FIG. 7C). Subsequent culturing was in the presence of 1 nM of PTX B oligomer. For direct analysis of clinical isolates, monocyte-depleted PBMCs from HIV-1-infected patients were activated for three days with PHA and then co-cultured with similarly activated PBMCs from uninfected donors in the presence of the indicated concentrations of PTX B oligomer (FIG. 7D). Virus replication was assayed in triplicate cultures, and error bars show standard deviation of the mean. A representative experiment is shown out of the three experiments performed with cells from different donors or patients. The uptake of [³H] thymidine was measured in triplicate long-term cultures of uninfected PBMC treated with indicated concentrations of PTX B oligomer (FIG. 7E). Error bars show standard deviation of the mean. In parallel, the number of cells in each culture (inset in FIG. 7E) is shown.

[0068] As would be predicted from the entry studies (FIG. 6), PTX B oligomer at 1 mM concentration inhibited replication of a primary R5 strain, 92US660 (FIG. 7A). Unexpectedly, a similar level of inhibition was also observed with the primary 92UG21 strain (FIG. 7B), and cell line-adapted X4 strains (LAI, panel C), entry of which was not affected by PTX B oligomer (FIG. 6). Thus, in addition to its effect on HIV-1 entry via the CCR5 receptor, PTX B oligomer exerted its anti-HIV activity via another mechanism that works at the post-entry step of viral replication, and did not depend on the identity of chemokine receptor used by the virus. This post-entry inhibitory effect of PTX B oligomer depended on the multiplicity of infection and was overcome when high virus inoculum was used. As in the above cell entry studies, neither R5 (92US660) nor X4 (LAI) strain replication was inhibited in PM1 cells (T-cell line; insets on FIGS. 7A and 7C).

[0069]FIG. 7 also shows that PTX B oligomer inhibited replication of uncloned primary HIV-1. The anti-HIV activity of PTX B oligomer on replication of uncloned primary HIV-1 was investigated by co-culturing uninfected donor PBMCs with activated PBMCs from HIV-1-infected patients. The number of infected cells in the PBMCs from such patients is usually low (Bukrinsky et al., Science 254:423-427, 1991), so the measured virus output in co-cultures is primarily a result of virus spread to new targets. A dose-dependent inhibition of virus replication by PTX B oligomer was observed (FIG. 7D). This effect was reproduced with PBMCs from three different patients; in all cases 1 nM of PTX B oligomer completely inhibited virus replication.

[0070] The foregoing inhibition of viral replication was not due to cytotoxic activity of PTX B oligomer. The uptake of [³H] thymidine and the change in cell numbers was measured in long-term cultures treated with PTX B oligomer. As shown in FIG. 7E, no significant difference in thymidine uptake between PTX B oligomer-treated and untreated cultures were observed during the entire course of the experiment. In fact, treated cultures had a slightly higher proliferation rate (FIG. 7E, inset), consistent with the well-known mitogenic effect of PTX B oligomer (Wong et al., Can. J. Physiol. Pharmacol. 74:559-564, 1996).

[0071] Therefore, the inhibitory effect of PTX B oligomer was exerted at both entry (for R5 strains) and post-entry (for both R5 and X4 viruses) steps of HIV-1 infection, and PTX B oligomer inhibited the replication of a wide variety of primary and cell line-adapted isolates.

EXAMPLE 8

[0072] This example illustrates that the inhibitory activity of the PTX B oligomer on HIV-1 entry is a signal-mediated process that does not involve blocking of HIV-1 binding to its receptors. FIG. 8 shows binding studies that were performed with [¹²⁵I] MIP-1β and gp120 to test whether the mechanism by which PTX B oligomer specifically inhibited entry of R5, but not of X4, HIV-1 strains was by interfering with binding of the virus; either directly (by blocking the HIV-1-binding site on CCR5) or indirectly (by changing conformation of CCR5).

[0073] Methods; binding studies. For each binding reaction, 1×10⁶ cells resuspended in binding buffer (50 mM Hepes, pH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA) were mixed with PTX B oligomer (1 nM), incubated for 10 min. at 37° C., and then transferred to ice. 550 pM [¹²⁵I]MIP-1β (specific activity 2200 Ci/mmol) was added to cells in the presence or absence of a 1000-fold molar excess of unlabeled MIP-1β, or 0.5 μg/ml of gp120, and reactions were incubated at 4° C. for 4 hr on a horizontal shaker. After washing with binding buffer containing 0.5 M NaCl, cell-bound radioactivity was counted in a gamma-counter. Binding was also measured in the presence of either a 1000-fold molar excess of unlabeled (cold) MIP-1β, or 0.5 μg/ml of gp120 of an R5 (JR-FL) or X4 (LAI) HIV-1 strain (FIG. 8A). To control for non-specific effects of the PTX B oligomer, binding was also analyzed using cells treated either with PTX B oligomer (1 nM) at 4° C., or with 75 ng/ml of BSA at 37° C. (FIG. 8B). Results are presented as means of duplicate samples, and the error bars show the deviation from the mean for each duplicate.

[0074] Results. FIG. 8A shows that binding of MIP-1β to PBMCs was competed by recombinant gp120 of an R5 strain HIV-1_(JR-FL), but not by gp120 of an X4 strain HIV-1_(LAI). Although treatment of the cells with PTX B oligomer increased [¹²⁵I] MIP-1β binding 3-fold, binding of this ligand was still effectively competed by gp120_(JR-FL) (FIG. 8A). To exclude the possibility that PTX B oligomer non-specifically affected binding of CCR5 ligands, for instance by acting as a bridge between the ligand and the cell, analysis was also performed using cells treated for 10 min. either with PTX B oligomer at 4° C. (to block signaling from B-oligomer receptor), or with BSA at 37° C. (to mimic the protein concentration in the experimental samples). Both treatments had no effect on [¹²⁵I] MIP-1β binding, compared to untreated samples (FIG. 8B).

[0075] Therefore, the inhibitory activity of the PTX B oligomer on HIV-1 entry via the CCR5 receptor is a signal-mediated process, which does not involve blocking of HIV-1 binding to CCR5. This result also indicates that binding of gp120 to CD4 and CCR5 is not sufficient to induce fusion of the viral and cellular membranes.

EXAMPLE 9

[0076] This example illustrates that PTX B oligomer blocks CCR5 signal transduction initiated by MIP-1β (FIG. 9A) and RANTES, but not SDF-1 α-initiated CCXR4 signaling (FIG. 9B), and demonstrates cross-desensitization of chemokine and PTX receptors. The foregoing Examples indicate that a post-binding step of virus entry is inhibited in PTX B oligomer-treated cells, and that this effect could be mediated by PTX B oligomer-induced alteration of an intracellular portion of the CCR5 molecule. This part of the receptor is involved in interactions with coupled G proteins, and Ca²⁺ mobilization. In response to natural ligands of CCR5 and CXCR4 (MIP-1β or RANTES, and SDF-1α, respectively), Ca²⁺ mobilization was measured after pre-treatment of the cells with PTX B oligomer (FIG. 9).

[0077] Methods; calcium mobilization assay. Assays were performed as described previously (Sherry et al., Proc. Natl. Acad. Sci. USA 95:1758-1763, 1998). In brief, 0.6 ml of Fura-2-loaded cells (5×10⁶ cells/ml) was transferred to an acrylic cuvette and stimulated with PTX B oligomer (500 ng/sample), SDF-1α (100 ng/sample), or MIP-1β (500 ng/sample). Fluorescence emission at 340 nm and 380 nm was measured on a Perkin-Elmer Luminescence Spectrometer LS50B.

[0078] Results. To measure Ca²⁺ mobilization, Fura-2-loaded PBMC were stimulated with 0.83 μg/ml of PTX B oligomer (B-ol.), (FIGS. 9A, 9B), 1.66 μg/ml of MIP-1β (FIG. 9C), or 0.1 μg/ml of SDF-1α (FIG. 9D) at the time indicated by the left arrow. At the time indicated by the right arrow (3-5 min. later), cells were rechallenged with PTX B oligomer (FIGS. 9C, 9D), MIP-1β (FIG. 9A), or SDF-1α (FIG. 9B) at the same concentration as in the first challenge. FIG. 9 shows the results of one representative experiment out of three performed with cells from different donors. These data are presented as the ratio of fluorescence emissions at 340 nm and 380 nm (F₃₄₀/F₃₈₀) over time.

[0079] Treatment of the cells with B-oligomer blocked Ca²⁺ flux initiated by MIP-1β (FIG. 9A) and RANTES, but not by SDF-1α (FIG. 9B). Pre-treatment of the cells with MIP-1β desensitized the response to B-oligomer (FIG. 9C), while pre-treatment with SDF-1α induced only partial desensitization (FIG. 9D). PTX B oligomer, at the suboptimal concentration used in these experiments (2 nM), did not desensitize its own receptor. The use of higher concentrations of PTX B oligomer for desensitization studies was hampered by a very slow return of the Ca²⁺ response to the baseline (more than 30 min.), thus precluding analysis by the Fura-2-based technique due to leaking of the dye from the cells.

[0080] Without being bound by theory, this pattern of Ca²⁺ response to sequential treatment of cells with PTX B oligomer and MIP-1β can be described by the phenomenon of heterologous desensitization. This desensitization is most likely attributable to signaling from a PTX B oligomer receptor, similar to the heterologous desensitization of CXCR1 and CXCR2 chemokine receptors, by signaling from opiate receptors (Grimm et al., J. Exp. Med. 188:317-325, 1998). While the possible binding of PTX B oligomer to CCR5 cannot formally be ruled out, several considerations make this scenario unlikely. First, PTX B oligomer did not compete with MIP-1β or gp120 for binding to CCR5 (FIG. 8). Second, PTX B oligomer did not induce down-regulation of CCR5, as evidenced by flow cytometric analysis. Third, PTX B oligomer did not induce Ca²⁺ flux and did not affect HIV-1 replication (FIG. 7) in PM1 cells, indicating that the anti-HIV activity of B-oligomer required signaling through its own receptor.

[0081] Therefore, post-binding CCR5 signaling, but not that of CXCR4, was blocked by PTX B oligomer and PTX and CCR5 receptors were cross-desensitized.

EXAMPLE 10

[0082] This example illustrates that PTX B oligomer inhibition of HIV-1 entry and signal transduction via CCR5 was mediated by protein kinase C (PKC) activity; and that the effect of PTX B oligomer on CCR5 was reversed by PKC inhibitors. FIG. 10A (panel A) shows that Ro-31-8220 reversed the inhibitory effect of PTX B oligomer on entry of R5 strains. PBMC cultures were either left untreated (control) or treated with PTX B oligomer (1 nM for 10 min.) with or without Ro-31-8220 (pre-treatment for 20 min. with 10 nM of the inhibitor). Entry of R5 (92US660) or X4 (LAI) strains of HIV-1 was measured as described in the description of FIG. 6. Results are presented as the mean of two independent experiments with cells from the same donor. The error bars show the deviation from the mean for duplicate samples. Similar results were obtained with cells from a different donor.

[0083]FIG. 10B (panel A) shows that Ro-31-8220 also reversed the inhibitory effect of PTX B oligomer on signaling from CCR5. PBMC cultures were either treated with PTX B oligomer (1 nM) alone or in combination with Ro-31-8220 (10 nM) (FIG. 10B, panels A and C) or left untreated (control) or treated with Ro-31-8220 (100 nM) alone (FIG. 10B, panels B and D) and stimulated with 500 ng/ml of RANTES (FIG. 10B, panels A and B) or 100 ng/ml of SDF-1α (FIG. 10B, panels C and D) at the time indicated by the arrow. Ca²⁺ flux was measured as described in Examples 8 and 9.

[0084] On its own, Ro-31-8220 did not significantly alter either of the above activities of CCR5 (FIG. 10A and 10B, panel B). Ro-31-8220 also did not act upon entry of X4 HIV-1 (FIG. 10A, panel B) or signaling by SDF-1α (FIG. 10B, panels C and D), that are mediated by CXCR4 and are not affected by PTX B oligomer. A similar result was observed with a different PKC inhibitor, Gö 6979. Taken together, these results indicate that PKC is involved in signal transduction from PTX B oligomer (and PTX) receptor to CCR5.

EXAMPLE 11

[0085] This example illustrates that pretreatment of cells with B-oligomer prevents capping of CCR5 HIV-1 receptors, indicating that signaling from chemokine receptors induced a major actin-dependent rearrangement of the cellular membrane. Receptor capping was used as an indicator of HIV-1-induced signaling.

[0086] Methods; immunofluorescent microscopy. Cells were treated with B-oligomer (1 nM) for 10 min. and then were inoculated with heat-inactivated HIV-1 (2×10⁶ cpm/ml of RT activity) or with 5 μg/ml of recombinant gp120_(JR-FL) (Progenics Pharmaceuticals, Tarrytown, N.Y.) or gp120_(LAI) (Intracel, Issaquah, Wash.). After 60 min. at 37° C. cells were washed and fixed in 4% buffered formaldehyde. After washing, cells were incubated for 20 min. in 1% FCS/PBS at room temperature, and 2×10⁵ cells were incubated for 30 min. at room temperature in 0.1 ml of 1% FCS/PBS with anti-CCR5 or anti-CXCR4 monoclonal antibody (2D7 and 12G5, respectively, obtained from Pharmingen, San Diego, Calif.; both antibodies neutralize HIV-1 infection), followed by a 30 min incubation with a secondary rhodamine-labeled anti-mouse IgG. After washing, cells were incubated with FITC-labeled anti-CD4 Mab (13B8.2, Immunotech, Westbrook, Me.; this antibody does not compete with gp120 for CD4 binding), then washed, spotted on slides, dried, and analyzed on an immunofluorescent imaging system using a dedicated software (MetaMorph, Universal Imaging Corporation, West Chester, Pa.).

[0087] Results. FIG. 11 shows an analysis of receptor capping by immunofluorescent microscopy. PBMCs were treated with 75 ng/ml of B-oligomer for 10 min. or left untreated, and receptor capping was analyzed by a dual-color immunofluorescent microscopy after inoculation with R5 HIV-1_(92US660) (FIG. 11A) or X4 HIV-1_(LAI) (FIG. 11B) viruses. CD4 was revealed with FITC-conjugated anti-CD4 mAb (thus producing green fluorescence), while chemokine receptors were stained with unlabeled primary and rhodamine-labeled secondary antibody (red fluorescence). Co-localization of receptors resulted in overlapping of red and green fluorescence, thus producing a yellow color. Approximately 50% and 30% of CD4/CCR5 and CD4/CXCR4 double-positive cells, respectively, exhibited capping.

[0088] Inoculation of PBMC cultures with either R5 (FIG. 11A) or X4 (FIG. 11B) HIV-1 strains, or their corresponding gp120s (Table 1), induced a characteristic co-capping of chemokine receptors with CD4. The fact that capped CCR5 and CXCR4 was detected using antibodies that compete with gp120 for chemokine receptor binding indicates that the observed signal came from free receptors that co-capped together with gp120-occupied receptors. Such interpretation is consistent with previously observed gp120-induced association of CD4 with several surface molecules, such as CD3, CD45RA, CD26, and HLA class I (Dianzani et al., Eur. J. Immunol. 25:1306-1311, 1995; Feito et al., Int. Immunol. 9:1141-1147, 1997), with RANTES-induced co-localization of CXCR4 and CD4 (Kinter et al., Proc. Natl. Acad. Sci. USA 95:11880-11885, 1998), and with co-capping of CCR2 and CCR5 in response to MCP-1 or RANTES (Nieto et al., J. Exp. Med. 186:153-158, 1997).

[0089] Pre-treatment of the cells with B-oligomer for 10 min. prevented capping of HIV-1 receptors, but only when capping was induced by R5 HIV-1 (FIG. 11A). This result indicates that capping was mediated by HIV-1-induced signaling from chemokine receptors, rather than from CD4, and that treatment with B-oligomer blocked this signal.

[0090] The effect of PTX B oligomer on capping induced by SDF-1α and MIP-1β was also analyzed (Table 1). This analysis was hampered by rapid down-regulation of CXCR4 or CCR5 after stimulation with SDF-1α or MIP-1β, respectively. Still, the results presented in Table 1 demonstrate that B-oligomer blocked capping induced by MIP-1β, but not by SDF-1α. Similar to HIV-1-or gp120-induced polarization, the capping involved not only the ligand-specific receptor, but also other molecules, including CD4.

[0091] These results indicate that signaling from chemokine receptors induces a major actin-dependent rearrangement of cellular membrane. TABLE 1 Quantification of receptor capping. CD4/CCR5³ CD4/CXCR4⁴ CCR5/CXCR4⁵ CCR5¹ CXCR4² cont. B-ol. cont. B-ol. cont. B-ol. MIP-1β 61 15 —⁶ — — — 40  9 HIV-1_(92US660) — — 53 10 — — — — gp120_(JR-FL) — — 66 — 14 — 39  3 SDF-1α 34 27 34 33 12  9 17 15 HIV-1_(LAI) — — — — 30 26 — — gp120_(LAI) — — 50 — 15 — 15 20

[0092] Cells were treated with PTX B oligomer (1 nM) for 10 min. or left untreated, and then incubated with the indicated stimulant for 60 min. Capping was assayed by immunofluorescent microscopy (see FIG. 11). Approximately 200 cells were counted for each treatment. For some treatments, experiment was repeated 2-3 times with similar results.

[0093] In summary, the Examples of the present invention show that PTX holotoxin as well as its binding subunit, B-oligomer, that lacks G_(i)-inhibitory activity, blocked entry of R5 but not of X4 strains into primary T lymphocytes. B-oligomer inhibited virus production by PBMC cultures infected with either R5 or X4 strains, indicating that B-oligomer can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to MIP-1β or RANTES; however, cell-surface expression of CCR5 and binding of MIP-1β or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity was mediated by signaling events that involved PKC. B-oligomer also blocked co-capping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect co-capping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. Examples of the present invention indicate that the PTX B oligomer cross-deactivated CCR5 and impaired its function as a co-receptor for HIV-1.

[0094] The present invention is applicable to a variety of physiological and pathological processes. This is because CCR5 is a receptor for the chemokines RANTES, MIP-1alpha, and MIP-1beta. These chemokines are involved in a variety of physiological and pathological processes associated with chemotactic responses, including chemotactic activity of cells during embryogenesis, chemotaxis of immune cells during immune responses, chemotaxis during inflammatory reactions in viral and bacterial infections, progressive neurological disorders (e.g., HTLV-associated myelopathy, multiple sclerosis; Calabresi et al., J. Neurovirol. 5:102-108), inflammation, and bacterial infections. 

We claim:
 1. A method for treating viral infections, comprising administering an effective amount of B. pertussis toxin (PTX) B oligomer to a patient having a viral infection, wherein the PTX B oligomer comprises from two to ten subunits of PTX selected from the group consisting of S2, S3, S4, S5, and combinations thereof, and wherein the viral infection is inhibited by a mechanism selected from the group consisting of inhibition of viral entry into the cell, inhibition of post-entry replication, inhibition of co-capping of a viral receptor or co-receptor, desensitization of a viral receptor or co-receptor, modulation of protein kinase C (PKC) activity, and combinations thereof.
 2. The method of claim 1 wherein the mechanism of inhibition of the viral infection is inhibition of viral entry into the cell.
 3. The method of claim 2 wherein the dose of PTX B oligomer administered each day is from about 0.01 mg/kg to about 500 mg/kg.
 4. The method of claim 2 wherein the viral infection is caused by HIV.
 5. The method of claim 2 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, 1S2-1S3-2S4, and 1S2-1S4-1S3-1S4-1S5.
 6. The method of claim 2 wherein viral entry is mediated by the chemokine receptor CCR5.
 7. The method of claim 1 wherein the mechanism of inhibition of the viral infection is inhibition of co-capping of a viral receptor or co-receptor.
 8. The method of claim 1 wherein the mechanism of inhibition of the viral infection is inhibition of post-entry replication.
 9. The method of claim 7 wherein the dose of PTX B oligomer administered each day is from about 0.01 mg/kg to about 500 mg/kg.
 10. The method of claim 7 wherein the viral infection is caused by HIV.
 11. The method of claim 7 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 12. The method of claim 7 wherein co-capping involves the chemokine receptor CCR5.
 13. The method of claim 1 wherein the mechanism of inhibition of the viral infection is desensitization of a viral receptor or co-receptor.
 14. The method of claim 13 wherein the dose of PTX B oligomer administered each day is from about 0.01 mg/kg to about 500 mg/kg.
 15. The method of claim 13 wherein the viral infection is caused by HIV.
 16. The method of claim 13 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 17. The method of claim 13 wherein the chemokine receptor CCR5 is desensitized.
 18. The method of claim 1 wherein the mechanism of inhibition of the viral infection is modulation of protein kinase C (PKC) activity.
 19. The method of claim 18 wherein the dose of PTX B oligomer administered each day is from about 0.01 mg/kg to about 500 mg/kg.
 20. The method of claim 18 wherein the viral infection is caused by HIV.
 21. The method of claim 18 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 22. The method of claim 18 wherein the chemokine receptor CCR5 is desensitized by modulation of protein kinase C (PKC) activity.
 23. A method for decreasing the infectivity of a cell or susceptibility of a cell to retroviral infection, wherein the cell expresses a CCR5 chemokine receptor, and wherein a virus uses the CCR5 chemokine receptor as a co-receptor, comprising contacting the cell with an amount of B. pertussis toxin (PTX) B oligomer sufficient to induce an antiviral mechanism selected from the group consisting of inhibition of viral entry into the cell, inhibition of post-entry replication, inhibition of co-capping of a viral receptor or co-receptor, desensitization of a viral receptor or co-receptor, modulation of protein kinase C (PKC) activity, and combinations thereof.
 24. A method for treating a CCR5 receptor-related physiological or pathological condition, comprising administering to a patient having the CCR5 receptor-related physiological or pathological condition, an amount of B. pertussis (PTX) B oligomer sufficient to desensitize the CCR5 receptor, wherein the PTX B oligomer is composed of from two to ten subunits of PTX selected from the group consisting of S2, S3, S4, S5, and combinations thereof.
 25. The method of claim 24, wherein the CCR5 receptor-related physiological or pathological condition is selected from the group consisting of progressive neurological disorders, HTLV-associated myelopathy, multiple sclerosis, inflammation, and infection.
 26. The method of claim 24 wherein the dose of PTX B oligomer administered each day is from about 0.01 mg/kg to about 500 mg/kg.
 27. A vaccine formulation for vaccinating against opportunistic infections from non-HIV pathogens in HIV-infected individuals, consisting essentially of an HIV-suppressing formulation of B. pertussis toxin (PTX) B oligomer (PTX B oligomer), an antigenic component specific for the non-HIV pathogen, with the proviso that the non-HIV pathogen is not influenza, and a vaccine-acceptable carrier.
 28. The vaccine formulation of claim 27 wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 29. The vaccine formulation of claim 27 wherein the pathogen is selected from the group consisting of Pneumocistis, Candida, CMV (Cytomegalovirus), Hepatitis virus (A, B and C), Pneumococcus, Mycobacterium tuberculosis, Mycobacterium aviium, Cryptosporidium, and Aspergillis.
 30. The vaccine formulation of claim 27 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 31. A method for vaccinating against opportunistic infections from non-HIV pathogens in HIV-infected individuals, comprising administering an HIV-suppressing formulation of B. pertussis toxin (PTX) B oligomer (PTX B oligomer) and an antigenic component specific for the non-HIV pathogen.
 32. The method of claim 31 wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 33. The method of claim 31 wherein the pathogen is of bacterial, viral, fungal, parasitic, or mycobacterial origin.
 34. The method of claim 33 wherein the pathogen is selected from the group consisting of Pneumocistis, Candida, CMV (Cytomegalovirus), Hepatitis virus (A, B and C), Pneumococcus, Mycobacterium tuberculosis, Mycobacterium aviium, Cryptosporidium, and Aspergillis.
 35. The method of claim 31 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 36. A method for vaccinating to protect an individual against HIV infection in an individual not infected with HIV comprising administering a vaccine composition comprising B. pertussis toxin (PTX) B oligomer (PTX B oligomer) and an antigenic component specific for HIV.
 37. The method of claim 36 wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 38. An HIV preventive vaccine formulation consisting essentially of B. pertussis toxin B oligomer (PTX B oligomer), an antigenic component specific for HIV, and a vaccine-acceptable carrier.
 39. The HIV preventive vaccine formulation of claim 38, wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 40. The HIV preventive vaccine formulation of claim 38, wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 41. A method for treating HIV infection, comprising administering B. pertussis toxin B oligomer (PTX B oligomer) in combination with an anti-HIV agent, wherein the HIV agent is selected from the group consisting of protease inhibitors, reverse transcriptase inhibitors, nuclear localization importation inhibitors, and combinations thereof.
 42. The method for treating HIV infection of claim 41 wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 43. The method for treating HIV infection of claim 41 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 44. A method for preventing HIV infection in an individual recently having transmissable contact with an HIV-infected individual, comprising administering B. pertussis toxin (PTX) B oligomer (PTX B oligomer) in combination with an agent selected from the group consisting a nuclear localization importation inhibitor, reverse transcriptase inhibitors, protease inhibitors, and combinations thereof.
 45. The method for preventing HIV infection in an individual recently having transmissable contact with an HIV infected individual of claim 44 wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 46. The method for preventing HIV infection in an individual recently having transmissable contact with an HIV infected individual of claim 44 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4.
 47. A pharmaceutical composition for treating viral infections consisting essentially of PTX B oligomer and a pharmaceutically acceptable carrier.
 48. The pharmaceutical composition of claim 47 wherein the dose of PTX B oligomer administered is from about 0.01 mg/kg to about 500 mg/kg.
 49. The pharmaceutical composition of claim 47 wherein the PTX B oligomer is selected from the group consisting of 1S2-1S4, 1S3-1S4, 1S2-1S3-2S4-1S5, and 1S2-1S3-2S4. 